This invention provides methods for the preparation of antibody fragment-targeted liposomes (“immunoliposomes”), including lipid-tagged antibody fragment-targeted liposomes, methods for in vitro transfection using the immunoliposomes, and methods for systemic gene delivery in vivo. The liposomes of the present invention are useful for carrying out targeted gene delivery and efficient gene expression after systemic administration. The specificity of the delivery system is derived from the targeting antibody fragments.
An ideal therapeutic for cancer would be one that selectively targets a cellular pathway responsible for the tumor phenotype and which is nontoxic to normal cells. While cancer treatments involving gene therapy have substantial promise, there are many issues that need to be addressed before this promise can be realized. Perhaps foremost among the issues associated with macromolecular treatments is the efficient delivery of the therapeutic molecules to the site(s) in the body where they are needed. A variety of delivery systems (a.k.a. “vectors”) have been tried including viruses and liposomes. The ideal delivery vehicle would be one that could be systemically (as opposed to locally) administered and which would thereafter selectively target tumor cells wherever they occur in the body.
The infectivity that makes viruses attractive as delivery vectors also poses their greatest drawback. Consequently, a significant amount of attention has been directed towards non-viral vectors for the delivery of molecular therapeutics. The liposome approach offers a number of advantages over viral methodologies for gene delivery. Most significantly, since liposomes are not infectious agents capable of self-replication, they pose no risk of transmission to other individuals. Targeting cancer cells via liposomes can be achieved by modifying the liposomes so that they selectively deliver their contents to tumor cells. There now exists a significant knowledge base regarding specific molecules that reside on the exterior surfaces of certain cancer cells. Such cell surface molecules can be used to target liposomes to tumor cells, because the molecules that reside upon the exterior of tumor cells differ from those on normal cells.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference.
Current somatic gene therapy approaches employ either viral or non-viral vector systems. Many viral vectors allow high gene transfer efficiency but are deficient in certain areas (Ledley F D, et al. Human. Gene Ther. (1995) 6:1129-1144). Non-viral gene transfer vectors circumvent some of the problems associated with using viral vectors. Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems (Massing U, et al., Int. J. Clin. Pharmacol. Ther. (1997) 35:87-90). Features of cationic liposomes that make them versatile and attractive for DNA delivery include: simplicity of preparation; the ability to complex large amounts of DNA; versatility in use with any type and size of DNA or RNA; the ability to transfect many different types of cells including non-dividing cells; and lack of immunogenicity or biohazardous activity (Felgner P L, et al., Ann. NY Acad. Sci. (1995) 772:126-139; Lewis J G, et al., Proc. Natl. Acad. Sci. USA (1996) 93:3176-3181). More importantly from the perspective of human cancer therapy, cationic liposomes have been proven to be safe and efficient for in vivo gene delivery (Aoki K et al., Cancer Res. (1997) 55:3810-3816; Thierry A R, Proc. Natl. Acad. Sci. USA (1997) 92:9742-9746). More than thirty clinical trials are now underway using cationic liposomes for gene therapy (Zhang W et al., Adv. Pharmacology (1997) 32:289-333; RAC Committee Report Human Gene Therapy Protocols-December 1998), and liposomes for delivery of small molecule therapeutics (e.g., antifungal and conventional chemotherapeutic agents) are already on the market (Allen T M, et al., Drugs (1997) 54 Suppl 4:8-14).
The transfection efficiency of cationic liposomes can be dramatically increased when they bear a ligand recognized by a cell surface receptor. Receptor-mediated endocytosis represents a highly efficient internalization pathway present in eukaryotic surface (Cristiano R J, et al., Cancer Gene Ther. (1996) 3:49-57, Cheng P W, Hum. Gene Ther. (1996) 7:275-282). The presence of a ligand on a liposome facilitates the entry of DNA into cells through initial binding of ligand by its receptor on the cell surface followed by internalization of the bound complex. A variety of ligands have been examined for their liposome-targeting ability, including transferrin and folate (Lee R J, et al., J. Biol. Chem. (1996) 271:8481-8487). Transferrin receptors (TfR) levels are elevated in various types of cancer cells including prostate cancers, even those prostate cell lines derived from human lymph node and bone metastases (Keer H N et al., J. Urol. (1990) 143:381-385); Chackal-Roy M et al., J. Clin. Invest. (1989) 84:43-50; Rossi M C, et al., Proc. Natl. Acad. Sci. USA (1992) 89:6197-6201; Grayhack J T. et al . . . J. Urol. (1979) 121:295-299). Elevated TfR levels also correlate with the aggressive or proliferative ability of tumor cells (Elliot R L, et al., Ann. NY Acad. Sci. (1993) 698:159-166). Therefore, TfR levels are considered to be useful as a prognostic tumor marker, and TfR is a potential target for drug delivery in the therapy of malignant cells (Miyamoto T, et al., Int. J. Oral Maxillofac. Surg. (1994) 23:430-433, Thorstensen K, et al. Scand. J. Clin. Lab. Invest. Suppl. (1993) 215:113-120). In our laboratory, we have prepared transferrin-complexed cationic liposomes with tumor cell transfection efficiencies in SCCHN of 60%-70%, as compared to only 5-20% by cationic liposomes without ligand (Xu L. et al., Hum. Gene Ther. (1997) 8:467-475).
In addition to the use of ligands that are recognized by receptors on tumor cells, specific antibodies can also be attached to the liposome surface (Allen T M et al., (1995) Stealth Liposomes, pp. 233-244) enabling them to be directed to specific tumor surface antigens (including but not limited to receptors) (Allen T M, Biochim. Biophys. Acta (1995) 1237:99-108). These “immunoliposomes,” especially the sterically stabilized immunoliposomes, can deliver therapeutic drugs to a specific target cell population (Allen T M, et al., (1995) Stealth Liposomes, pp 233-244). Park, et al. (Park J W, et al., Proc. Nail. Acad. Sci. USA (1995) 92:1327-1331) found that anti-HER-2 monoclonal antibody (Mab) Fab fragments conjugated to liposomes could bind specifically to HER-2 overexpressing breast cancer cell line SK-BR-3. The immunoliposomes were found to be internalized efficiently by receptor-mediated endocytosis via the coated pit pathway and also possibly by membrane fusion. Moreover, the anchoring of anti-HER-2 Fab fragments enhanced their inhibitory effects. Doxorubicin-loaded anti-HER-2 immunoliposomes also showed significant and specific cytotoxicity against target cells in vitro and in vivo (Park J W, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1327-1331). In addition, Suzuki et al., (Suzuki S, et al., Br. J. Cancer (1997) 76:83-89) used an anti-transferrin receptor monoclonal antibody conjugated immunoliposome to deliver doxorubicin more effectively in human leukemia cells in vitro. Huwyler et al. (Huwyler J, et al., Proc. Natl. Acad. Sci. USA (1996) 93:14164-14169) used anti-TfR monoclonal antibody immunoliposome to deliver daunomycin to rat glioma (RT2) cells in vivo. This PEGylated immunoliposome resulted in a lower concentration of the drug in normal tissues and organs. These studies demonstrated the utility of immunoliposomes for tumor-targeting drug delivery. It should be noted that the immunoliposome complexes used by Suzuki et al. and Huwyler et al. differ from those of the invention described herein in that they are anionic liposomes and that the methods used by Suzuki et al. and Huwyler et al. are not capable of delivering nucleic acids.
Single-Chain Antibody Fragments
Progress in biotechnology has allowed the derivation of specific recognition domains from Mab (Poon R Y, (1997) Biotechnology International: International Developments in the Biotechnology Industry, pp. 113-128). The recombination of the variable regions of heavy and light chains and their integration into a single polypeptide provides the possibility of employing single-chain antibody derivatives (designated scFv) for targeting purposes. Retroviral vectors engineered to display scFv directed against carcinoembryonic antigen, HER-2, CD34, melanoma associated antigen and transferrin receptor have been developed (Jiang A, et al., J. Virol. (1998) 72:10148-10156, Konishi H. et al., Hum. Gene Ther. (1994) 9:235-248, Martin F, et al., Hum. Gene Ther. (1998) 9:737-746). These scFv directed viruses have been shown to target, bind to and infect specifically the cell types expressing the particular antigen. Moreover, at least in the case of the carcinoembryonic antigen, scFv was shown to have the same cellular specificity as the parental antibody (Nicholson I C, Mol. Immunol. (1997) 34:1157-1165).
The combination of cationic liposome-gene transfer and immunoliposome techniques appears to be a promising system for targeted gene delivery.